Multidimensional narrow bore liquid chromatography analysis of Ro 24-0238 in human plasma.

A highly sensitive LC method has been developed and validated for quantitation of Ro 24-0238 in human plasma using Ro 24-2446 as an internal standard. With 1 ml of plasma, the limit of quantitation of the method was 50 pg ml-1 of Ro 24-0238. After solid-phase extraction with C18 reversed-phase cartridges, the samples were reconstituted in an acidic buffer solution; under these conditions, Ro 24-0238 and Ro 24-2446 (IS) were converted to their cationic forms. The LC system employed a strong cation exchange column and a narrow bore reversed-phase column, connected via a column switching valve. The cationic analyte and internal standard were separated from most of the endogenous components of plasma on the cation exchange column. A small fraction containing the analyte and the internal standard was transferred by automated valve switching to the narrow bore reversed-phase column, which further resolved the individual components. The chromatography was monitored by UV absorption at 322 nm. The overall intra-assay precision was 3.6% (RSD) and the per cent error was less than +/- 11%. The overall inter-assay precision was 3.9% (RSD). Linearity was demonstrated in a concentration range of 50-5000 pg ml-1. This method has been applied to pharmacokinetic studies of Ro 24-0238 in man.

Determination of a new antibacterial agent (Ro 23-9424) by multidimensional high-performance liquid chromatography with ultraviolet detection and direct plasma injection.

Analysis of a new antibacterial agent, Ro 23-9424 (I), in plasma has been complicated by the fact that its metabolite, fleroxacin (II), is formed not only in vivo, but also nonenzymatically by the hydrolysis of the ester bond of I. In order to minimize sample preparation time and possible hydrolysis during sample preparation, a high-performance liquid chromatographic procedure was developed which features direct injection of plasma and multidimensional chromatography. The first dimension size-exclusion separation allows plasma proteins to elute with the column void volume. The second dimension reversed-phase column provides a high-resolution separation dependent upon the hydrophobicity of the sample species. With a 5-microliters injection, the limit of quantitation of the method is 0.35 microgram/ml for I and 0.27 microgram/ml for II. The method was used to determine steady state plasma vs. time profiles for I and II from 750 mg i.v. doses of I administered twice daily.

Determination of Ro 23-7637 in dog plasma by multidimensional ion-exchange-reversed-phase high-performance liquid chromatography with ultraviolet detection.

Ro 23-7637 (I) is a new drug under development for the treatment of metabolic diseases. A high-performance liquid chromatographic-ultraviolet detection (HPLC-UV) analytical procedure for its analysis in dog plasma was developed and is reported here. Following C18 solid-phase extraction, the sample is applied to a strong cation-exchange column in the first dimension. The analyte and internal standard, Ro 24-4558 (II), are transferred to a base-deactivated C18 reversed-phase column in the second dimension (orthogonal separation mechanism), with UV detection at 254 nm. The reversed-phase solid-phase extraction provides a gross isolation of the drug, based on hydrophobicity. The first-dimension ion-exchange separation allows neutral species and anions to elute with the column void volume, while separating basic species according to pKa. The second dimension provides a high-resolution separation dependent upon the hydrophobicity of the sample species. The rationale for using orthogonal multidimensional chromatography was that an exhaustive examination of reversed-phase and normal-phase columns invariably resulted in co-elution of I with endogenous plasma components, which limited the sensitivity of the method. We have utilized C18 solid-phase extraction, followed by multidimensional HPLC-UV, to develop an accurate and precise analytical method whose limit of quantitation, 5 ng/ml using 0.5 ml of plasma, is determined by inherent detector sensitivity. Increased sensitivity can be readily achieved by using more sample or by using microbore HPLC on the second dimension.